ABSTRACT Sex differences in the cardiovascular response to alcoholism exist such that women are apparently more sensitive to the toxic effects of alcohol vs men, although the mechanisms which underlie this phenomenon are poorly understood and have not been systematically investigated. Greater vulnerability to alcoholic heart disease in women is exacerbated by evidence indicating that post-menopausal women have higher mortality rates after myocardial infarction vs aged men, and it is unclear whether the effects of aging and estrogen (E2) deficiency are additive with regard to ethanol-induced cardiac ischemia/reperfusion (I/R) injury. Because of the pivotal role played by the mitochondria in the maintenance of cell survival and cardioprotection, it is logical that ethanol and age-associated increases in I/R injury might arise from altered responses in the mitochondrial subproteome. To address this problem, we will employ in vitro isolated heart and in vivo mitochondrial respiration studies in conjunction with the newly emerging iTRAQ 8plex proteomics approach, LC MS/MS and PANTHER ontological analysis to characterize alterations in mitochondrial signaling occurring with aging and E2 deficiency in the female rat myocardium following chronic ethanol ingestion. Of particular interest are adaptive changes in proteins associated with metabolism, oxidative stress, and cell death. Specific aims are as follows: 1) chronic ethanol ingestion will exacerbate age-related increases in I/R injury in association with reduced mitochondrial targeting of known cardioprotective signals and estrogen receptor (ER) subtypes, and 2) chronic ethanol ingestion will exacerbate the effects of E2 deficiency on age dependent disruptions in protective mitochondrial signaling. Infarct size and apoptotic cell death will be assessed in Langendorff-perfused hearts following global I/R in adult and aged ovary-intact and gonadectomized female rats;respiration studies will be performed in isolated mitochondria. Western blotting and qRT- PCR will be employed for mitochondrial ER subtype targeting and downstream signals;iTRAQ and multiple reaction monitoring (MRM) for protein changes and post-translational modifications of ERs. The high throughput, targeted iTRAQ proteomics approach we describe here will allow us to provide critical and novel mechanistic insight, in a relatively short time, on the role of female sex in the development of alcohol related cardiac dysfunction in aged and adult rats. Moreover, information gleaned from the proposed studies should provide strong, direct support for new therapeutic targets for treating alcoholic heart disease in postmenopausal women, as well as provide impetus and rationale for the design of future experimental studies. PUBLIC HEALTH RELEVANCE: Current experimental evidence supports a J- or U-shaped association between alcohol consumption and heart attack and/or high blood pressure. As such, while light-to-moderate drinking has been associated with protection from heart disease, heavy drinking is associated with the development of heart failure, which remains a major public health crisis and a leading cause of morbidity and mortality in aged men and women. The CDC has most recently estimated that ~75,000 deaths per year can be attributed to the harmful effects of alcohol. Moreover, nearly 33% of chronic alcohol-dependent individuals exhibit cardiac dysfunction, which often becomes manifest as an alcohol-specific heart failure. Interestingly, women appear to more sensitive to the toxic effects of alcohol on the heart when compared to men, even though total alcohol consumption appears to be less in alcoholic women vs men. It is also important to note that heart disease remains the leading cause of death in post-menopausal women, with mortality rates surpassing those of age-matched men. The specific mechanisms which produce this gender-specific cardiac dysfunction and enhanced susceptibility to ischemic injury in the aged female heart following chronic ethanol ingestion have not been directly examined, and the studies proposed herein will fill this information gap. We propose a comprehensive and novel approach that will identify proteins that comprise the functional sequelae of complex cardiac phenotypes of chronic ethanol ingestion, and will further characterize possible targets for therapeutic intervention in the aged female population.